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Developmental Biology

检测果蝇黑色素的内分泌干扰的方法

Published: July 03, 2019
doi:
10.3791/59535
, , , , ,
1Institute of Biosciences and BioResources,National Research Council of Italy (CNR), 2Institute of Research on Terrestrial Ecosystems (IRET),National Research Council of Italy (CNR), 3Department of Biology,UniNa “Federico II”

Summary

内分泌干扰化学品 (EDC) 是生物体和自然环境的严重问题。果蝇黑色素是研究体内EDC效应的理想模型。在这里,我们提出的方法,以调查果蝇的内分泌中断,解决EDC对苍蝇的生育、生育、发育时间和寿命的影响。

Abstract

近年来,越来越多的证据表明,所有生物体和环境都暴露于激素样的化学物质,即内分泌干扰物(EDCs)。这些化学物质可能改变内分泌系统的正常平衡,导致不利影响,以及人类中荷尔蒙紊乱的数量增加或野生动物物种的生长和繁殖减少。对于某些 EDC,有记录的健康影响和限制其使用。然而,对于他们中的大多数来说,在这个意义上仍然没有科学证据。为了验证一种化学物质在全有机体中的潜在内分泌效应,我们需要在适当的模型系统中以及果蝇,果蝇,果蝇黑色素气。在这里,我们报告详细的体内协议,以研究果蝇的内分泌中断,解决EDC对苍蝇的繁殖/生育能力,发育时间和寿命的影响。在过去几年中,我们使用这些果蝇生命特性来研究暴露于17-β-苯乙醇(EE2)、双酚A(BPA)和双酚AF(BPA F)的影响。总之,这些测定涵盖了所有果蝇生命阶段,并使得评估所有激素介导过程中的内分泌中断成为可能。成熟/生育力和发育时序测定有助于分别测量EDC对苍蝇生殖性能和发育阶段的影响。最后,寿命测定涉及慢性EDC对成人的接触,并测量他们的存活率。然而,这些生命特征也可能受到几个必须仔细控制的实验性因素的影响。因此,在这项工作中,我们建议一系列程序,我们已经优化了这些检测的正确结果。这些方法允许科学家建立内分泌中断的任何EDC或混合不同的EDCs在果蝇,虽然要确定负责这种效果的内分泌机制,可能需要进一步的文章。

Introduction

人类活动已经释放出大量的化学物质,这对生物体和自然生态系统来说是一个严重的问题。在这些污染物中,估计约有1,000种不同的化学物质可能改变内分泌系统的正常平衡;根据这个特性,它们被归类为干扰内分泌的化学物质(EDCs)。具体来说,根据内分泌学会最近的定义,EDCs是”一种外源性化学物质,或化学品的混合物,可以干扰激素作用的任何方面”2 。在过去的三十年里,越来越多的科学证据表明,EDC会影响动植物的繁殖和发育3,4,5,6,7, 8.此外,EDC接触与一些人类疾病的日益流行有关,包括癌症、肥胖症、糖尿病、甲状腺疾病和行为障碍9、10、11。

EDC的一般机制

由于其分子特性,EDCs的行为类似于激素或激素前体3,4,5,6,7,8,9, 10,11,12.从这个意义上说,它们可以通过模仿激素活性或阻断内源性激素结合来与激素受体结合,扰乱内分泌系统。在第一种情况下,与受体结合后,它们可以像其天然激素那样激活它。在其他情况下,EDC与受体结合会阻止其天然激素的结合,因此受体被阻断,无法再被激活,即使存在其天然激素3。因此,EDC 可以影响多个过程,例如内源性激素的合成、分泌、运输、代谢或外围作用,这些过程负责维持的平衡、繁殖、发展和/或行为。有机体。受体绑定不是迄今为止EDC描述的唯一行动方式。现在很清楚,他们也可以通过在酶通路中招募联合激活剂或核心压榨器,或者通过修改表观遗传标记来解除基因表达10、11、12、13 ,14,不仅对当代人,而且对后代的健康带来影响。

果蝇激素

已广泛研究了选定的EDC的潜在影响,无论是在野生动物物种中,还是在内分泌机制相当广为人知的几个模型系统中。对于无脊椎动物,影响生长、发育和繁殖的内分泌系统在昆虫中被广泛特征,原因有若干原因,涉及它们在生物研究领域的广泛应用、其经济重要性和最后,开发能够专门干扰害虫激素系统的杀虫剂。

特别是在昆虫中,果蝇D.黑色素气仪已被证明是一个非常强大的模型系统,以评估EDCs的潜在内分泌效应。在黑色素和脊椎动物中,激素在整个生命周期中起着重要的作用。在这个有机体中,有三个主要的荷尔蒙系统,其中涉及类固醇激素20-羟基克西酮(20E)15,16,Sesquiterpenoid青少年激素(JH)17,和神经肽和肽/蛋白质激素18.第三组由最近发现但明显涉及各种生理和行为过程的肽组成,如长寿、平衡、代谢、生殖、记忆和运动控制。20E与胆固醇衍生的类固醇激素(如雌二醇)同源,而JH与视黄酸有一些相似之处;他们两者都是德罗索菲拉19,20中比较著名的激素。它们的平衡对于协调蜕皮和蜕变,以及控制几个发育后过程(如生殖、寿命和行为21)至关重要,从而为测试内分泌提供了不同的可能性在果蝇的中断。此外,异类固醇激素和JHs是所谓的第三代杀虫剂的主要目标,这种杀虫剂旨在干扰昆虫的发育和生殖内分泌介导过程。这些化学品的激动剂或拮抗作用模式是众所周知的,因此它们可以作为评估潜在EDCs对昆虫生长、繁殖和发育影响的参考标准22。例如,Methoprene已被广泛用于控制蚊子和其他水生昆虫23,24,作为JH激动剂,抑制20E诱导的基因转录和蜕变。

除了激素,果蝇中的核受体(NR)超级家族也是众所周知的;它包括18个进化保存转录因子,涉及控制激素依赖性发育途径,以及生殖和生理学25。这些激素NR属于所有六个NR超级家族亚型,包括那些涉及神经传递26,两个视黄酸NR,和那些类固醇NR,在脊椎动物,代表EDCs27的主要目标之一。

果蝇作为研究EDC的模型系统

目前,根据分子特性,世界各地的几个环境机构将干扰内分泌系统的可能性归因于不同的人造化学品。鉴于EDC是环境和生物体的全球性和普遍问题,这一领域的研究的总体目标是减轻其疾病负担,并保护生物免受其不利影响。为了加深对一种化学物质潜在的内分泌效应的理解,有必要在体内对其进行测试。为此,D. melanogaster表示一个有效的模型系统。迄今为止,果蝇已被广泛用作体内模型,以评估几种环境EDC的影响;据报道,暴露于几种EDCs,如二丁基邻苯二甲酸酯(DBP)28,双酚A(BPA),4-非基酚(4-NP),4-吨-乙基苯酚(4-t-OP)29,甲基对羟基苯甲酸酯(MP)30,乙基对羟基苯甲酸酯(EP)31, 32、二乙基(乙酰乙酰)邻苯二甲酸酯(DEHP)33和17-α-乙酸乙酯(EE2)34,影响脊椎动物模型中的新陈代谢和内分泌功能。有几个原因导致它作为这一研究领域的一个模型。除了对其内分泌系统的深入了解外,其他优势还包括其生命周期短、成本低、基因组易于操作、研究历史悠久以及多种技术可能性(参见 FlyBase 网站,http://flybase.org/)。D. melanogaster还为轻松研究跨代效应和种群对环境因素的反应提供了强大的模型,并避免了与高等动物体内研究相关的伦理问题。此外,果蝇与人类共享高度的基因保护,这可能使果蝇EDC检测有助于预测或建议这些化学物质对人类健康的潜在影响。除了扩大对人类健康影响的理解外,果蝇还有助于评估EDC暴露于环境中的风险,如生物多样性丧失和环境退化。最后,果蝇提供了在实验室使用的额外优势,在实验室中,可能影响其发育、繁殖和寿命的因素可以加以控制,以便将任何变异归因于要测试的物质。

考虑到这一点,我们优化了简单和强大的健身测定,以确定EDC对一些果蝇激素特性的影响,如生育/生育、发育时机和成人寿命。这些测定已广泛用于一些EDCs 23,24,25,26,27。特别是,我们使用以下协议来评估暴露于合成雌激素 EE234和 BPA 和双酚 AF (BPA F)(未发布数据)的影响。这些协议可以很容易地修改,以研究给定EDC在一次的影响,以及多个EDC在D.melanogaster的综合影响。

Protocol

1. 食物准备 对于库存维持和幼虫生长,使用含有3%粉状酵母、10%蔗糖、9%预煮玉米粉、0.4%琼脂的玉米粉培养基,其后称为玉米粉培养基(CM)。 将30克酵母放入100 mL的自来水中,煮沸,煮15分钟。 另外,将90克预先煮熟的玉米粉、100克糖和4克琼脂混合成900 mL的自来水。 将溶液煮沸,降低热量,连续煮5分钟。 5分钟后,加入热酵母溶液,再煮15分钟。 关?…

Representative Results

在本节中,以简化方案的形式报告上述协议的关键步骤。鉴于苍蝇倾向于避免令人不快的化合物,第一件事就是测定所选EDC的味道。这可以通过混合食品着色(例如,红色食品染料40号)35与食品补充与选定的EDC在各种剂量或与溶剂单独。以这些介质喂养的苍蝇在立体显微镜下接受检查,食物摄入量由腹部着色估算(图1)。图1显示了一个典型的?…

Discussion

果蝇D.melanogaster被广泛用作体内模型系统,以研究环境EDC的潜在影响,如DBP28,双酚A,4-NP,4-ter-OP29,MP30,EP31, 32, DEHP33,和 EE234.有几个原因导致它作为模型在这一研究领域使用。除了作为模型系统无可争议的优势外,果蝇与人类共享高度的基因保护,而果蝇EDC测定法可能…

Disclosures

The authors have nothing to disclose.

Acknowledgements

作者感谢奥索利娜·佩蒂洛的技术支持。作者感谢玛丽亚罗萨里亚·阿莱塔博士(CNR)对书目的支持。作者感谢古斯塔沃·达米诺·米塔博士将其介绍给EDC世界。作者感谢莱卡微系统公司和帕斯夸尔·罗曼诺的帮助。这项研究得到了项目PON03PE_001110_1的支持。”纳米技术定向学的Sviluppo di nanotecnoe a.rigenerazione e Ricostruzione Tissutale, 在奥东托里亚/奥多里亚/奥科利西亚的植入物 e 感理”acronimo”SORRISO”承诺:PO FESR 2014-2020 CAMPANIA;项目PO FESR坎帕尼亚2007-2013年”纳纳特诺诺诺诺托控制迪莫莱科尔生物-技术纳塔诺诺诺诺诺诺”。

Materials

17α-Ethinylestradiol Sigma E4876-1G
Agar for Drosophila medium BIOSIGMA 789148
Bisphenol A Sigma 239658-50G
Bisphenol AF Sigma 90477-100MG
Cornmeal CA' BIANCA
Diethyl ether Sigma
Drosophila Vials BIOSIGMA 789008 25×95 mm
Drosophila Vials BIOSIGMA 789009 29×95 mm
Drosophila Vials Kaltek 187 22X63
Embryo collection cage Crafts Plexiglass cylinder (12,5 x7 cm) with an open end and the other end closed by a rectangular base in which a slot allows the insertion of special trays for laying
Ethanol FLUKA 2860
Etherizer Crafts cylindrical glass container with a cotton plug
Glass Bottle 250mL Bottles
Glass Vials Microtech ST 10024 FLAT BOTTOM TUBE 100X24
Hand blender Pimmy Ariete food processor
Instant Success yeast ESKA Powdered yeast
Laying tray Crafts plexiglass trays (11 x 2,6 cm) in wich to pour medium for laying
Methyl4-hydroxybenzoate SIGMA H5501
Petri Dish Falcon 351016 60×5
Red dye no. 40 SIGMA 16035
Stereomicroscope with LED lights Leica S4E
Sucrose HIMEDIA MB025
Tomato sauce Cirio
DOWNLOAD MATERIALS LIST

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Drosophila MelanogasterEndocrine DisruptionFertilityDevelopmentLifespanEndocrine DisruptorsIn VivoReproductive PerformanceCornmeal MediumEE2Carbon Dioxide
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Bovier, T. F., Cavaliere, D., Colombo, M., Peluso, G., Giordano, E., Digilio, F. A. Methods to Test Endocrine Disruption in Drosophila melanogaster. J. Vis. Exp. (149), e59535, doi:10.3791/59535 (2019).
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